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J Biol Chem, Vol. 273, Issue 37, 24000-24008, September 11, 1998
From the Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow G12 8QQ Scotland, United Kingdom
The long isoform of the rat thyrotropin-releasing
hormone receptor (TRHR) was modified by the addition of a vesicular
stomatitis virus (VSV) epitope tag and green fluorescent protein (GFP).
VSV-TRHR-GFP bound TRH with affinity similar to that of the unmodified
receptor and stimulated [3H]inositol phosphate
production. A clone stably expressing VSV-TRHR-GFP at some 120,000 copies/cell was selected to visualize this receptor during cellular
exposure to TRH. Internalization was detected within 3-5 min after
treatment with 1 × 10
Real Time Visualization of Agonist-mediated Redistribution and
Internalization of a Green Fluorescent Protein-tagged Form of the
Thyrotropin-releasing Hormone Receptor
7 M TRH, with
dramatic reductions in plasma membrane localization achieved within
10-15 min. The TRHR antagonist/inverse agonist chlordiazepoxide
competitively inhibited internalization. Hyperosmotic sucrose inhibited
internalization of VSV-TRHR-GFP, measured both by intact cell
[3H]TRH binding studies and by confocal microscopy. Now
TRH caused a redistribution of VSV-TRHR-GFP to highly punctate but
plasma membrane-delineated foci. Pretreatment with the
microtubule-disrupting agent nocodazole allowed internalization of the
VSV-TRHR-GFP construct but only into vesicles that remained in close
apposition to the plasma membrane. Covisualization of VSV-TRHR-GFP and
Texas Red transferrin initially indicated entirely separate
localizations. After exposure to TRH substantial amounts of
VSV-TRHR-GFP were present in vesicles overlapping those containing
Texas Red transferrin. Such results demonstrate the G protein-coupling
capacity and provide real time visualization of the processes of
internalization of a TRH-receptor-GFP construct in response to
agonist.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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